Using the short-lived isotope 11C in mechanistic studies of photosynthate transport

Tracer techniques have been central in studies of transport in plants. In the case of carbon, the only readily available radioactive tracer has been C-14, although C-11 was used for a short time before C-14 could be made. Tracers have usually had to be measured by destructive harvesting of the plant, giving a practical limit to the data resolution in both time and space. A major advantage of the short-lived, positron-emitting tracers, of which C-11 is one example, is that in vivo measurement is possible, giving detailed time series of tracer data in many locations and opening up powerful new techniques of data analysis. Medical applications of these isotopes have utilised both dynamic imaging and time courses of uptake or washout. Unfortunately, few plant biology laboratories have realised the potential of these techniques, possibly because of the large physics infrastructure needed. In this paper we review the concepts behind the use of these short-lived tracers in plant physiology, and illustrate with several cases where C-11 was an essential tool

Direct measurement of sieve element hydrostatic pressure reveal strong regulation of sieve element hydrostatic pressure after pathway blockage

According to the Munch hypothesis, solution flow through the phloem is driven by a hydrostatic pressure gradient. At the source, a high hydrostatic pressure is generated in the collection phloem by active loading of solutes, which causes a concomitant passive flow of water, generating a high turgor pressure. At the sink, solute unloading from the phloem keeps the turgor pressure low, generating a source-to-sink hydrostatic pressure gradient. Localised changes in loading and unloading of solutes along the length of the transport phloem can compensate for small, short-term changes in phloem loading at the source, and thus, maintain phloem flow to the sink tissue. We tested directly the hydrostatic pressure regulation of the sieve tube by relating changes in sieve tube hydrostatic pressure to changes in solute flow through the sieve tube. A sudden phloem blockage was induced ( by localised chilling of a 1-cm length of stem tissue) while sieve-tube-sap osmotic pressure, sucrose concentration, hydrostatic pressure and flow of recent photosynthate were observed in vivo both upstream and downstream of the block. The results are discussed in relation to the Munch hypothesis of solution flow, sieve tube hydrostatic pressure regulation and the mechanism behind the cold-block phenomenon

Phloem hydrostatic pressure relates to solute loading rate: a direct test of Münch hypothesis

According to the Munch hypothesis, a flow of solution through the sieve tubes is driven by a hydrostatic pressure difference between the source ( or collection) phloem and the sink ( or release) phloem. A high hydrostatic pressure is maintained in the collection phloem by the active uptake of sugar and other solutes, with a concomitant inflow of water. A lower pressure is maintained in the release phloem through solute unloading. In this work we directly test the role of solute uptake in creating the hydrostatic pressure associated with phloem flow. Solute loading into the phloem of mature leaves of barley and sow thistle was reduced by replacing the air supply with nitrogen gas. Hydrostatic pressure in adjacent sieve elements was measured with a sieve-element pressure probe, a cell pressure probe glued to the exuding stylet of aphids that had been feeding from the phloem. Sieve element sap was sampled by aphid stylectomy; sap osmotic pressure was determined by picolitre osmometry and its sugar concentration by enzyme-linked fluorescence assays. Samples were taken with a time resolution of similar to 2 - 3 min. In accordance with Munch's proposal a drop in osmotic and hydrostatic pressure in the source phloem following treatment of the source leaf with N-2 was observed. A decrease in sugar concentration was the major contributor to the change in osmotic pressure. By observing these variables at a time resolution of minutes we have direct observation of the predictions of Munch

What can tracer profiles tell us about the mechanisms giving rise to them?

For a long time, tracer profiles observed at various positions along the phloem transport pathway have been used to infer details about both the mechanisms and the amount of flow through this pathway within whole plants. But the mechanisms still elude us. This paper investigates why this is so and shows that although the amount of mechanistic information available from tracer profiles is rather limited, they do provide reliable quantitative measurements of the amount of flow. Detailed quantitative analysis of tracer profiles in barley leaves is used to illustrate techniques of mechanistic interpretation from input-output analysis and indicates the need of two compartments to describe carbon flow through a leaf from fixation to phloem loading

Solute is imported to elongating root cells of barley as a pressure driven flow of solution

This work relates solute import to elongating root cells in barley to the water relations of the symplastic pathway under conditions of varied plant K+ status. K+ is a major constituent of phloem sieve element (SE) sap, and as an osmoticum, it is believed to have a role in maintaining SE hydrostatic pressure and thus sap flow from source to sink tissue. The hypothesis that the solute import to elongating root cells is linked to pressure driven flow from the sieve tube is examined.Plants were grown in solutions containing either 0.05 m M (low K) or 2.05 m M (high K) K+ concentration. Solute import to the root elongation zone was estimated from biomass accumulation over time accounting for respiration and root elongation rate. SE sap K+ concentration was measured using X-ray microanalyses and osmotic pressure by picolitre osmometry. SE hydrostatic pressure was measured directly with a pressure probe glued onto an excised aphid stylet. Elongating root cell hydrostatic pressure was measured using a cell pressure probe.The low-K plants had lower SE K+ concentration and SE hydrostatic pressure compared to the high-K plants, but the elongating root cell hydrostatic pressure was similar in both treatments, thus the pressure difference between the SE and elongating root cells was less in the low-K plants compared to the high-K plants.The solute import rate to elongating root cells was lower in the low K plants and the reduction could be accounted for as a pressure driven solute flux, with a reduction both in the pressure difference between root sieve elements and elongating cells, and in the sap concentration

Short-term storage of carbohydrate in stem tissue of apple (Malus domestica), a woody perennial: evidence for involvement of the apoplast

This work investigates the pathway and mechanism for lateral retrieval of carbohydrate into the transport phloem of apple stems ( Malus domestica Borkh.). A heat exchanger was set up on the stem, allowing rapid chilling and subsequent re-warming of stem segments while the time course of axial transport of C-11-labelled photoassimilate was measured at a position similar to 65 mm downstream of the heat exchanger. Whenever axial transport was blocked by a sudden chill at the heat exchanger, transport 65 mm downstream from the blockage immediately slowed but did not stop, showing that there was retrieval of solutes into the pathway ( buffering), within that 65 mm of stem, to help maintain the axial flow. Use of PCMBS, an inhibitor of sugar transporters, showed that the buffering included retrieval of sugar from the apoplast. We concluded that in apple, apoplastic sugar in stem tissue can buffer phloem transport during short-term changes in supply and demand for carbohydrates. Buffering was stronger when mobile reserves in the stem were higher, for example late in the photoperiod, or if carbohydrate demand in the terminal sink was increased. We also suggest that the concentration of sugars in the apoplast is a regulator of carbohydrate storage and re-mobilisation

Jasamonic acid treatment to part of the root system is consistent with simulated leaf herbivory, diverting recently assimilated carbon towards untreated roots within an hour

It is known that shoot application of jasmonic acid (JA) leads to an increased carbon export from leaves to stem and roots, and that root treatment with JA inhibits root growth. Using the radioisotope (11)C, we measured JA effects on carbon partitioning in sterile, split-root, barley plants. JA applied to one root half reduced carbon partitioning to the JA-treated tissue within minutes, whereas the untreated side showed a corresponding--but slower--increase. This response was not observed when instead of applying JA, the sink strength of one root half was reduced by cooling it: there was no enhanced partitioning to the untreated roots. The slower response in the JA-untreated roots, and the difference between the effect of JA and temperature, suggest that root JA treatment caused transduction of a signal from the treated roots to the shoot, leading to an increase in carbon allocation from the leaves to the untreated root tissue, as was indeed observed 10 min after the shoot application of JA. This supports the hypothesis that the response of some plant species to both leaf and root herbivores may be the diversion of resources to safer locations

Turgor, solute import and growth in maize roots treated with galactose

It has been observed that extension growth in maize roots is almost stopped by exposure to 5 mM D-galactose in the root medium, while the import of recent photoassimilate into the entire root system is temporarily promoted by the same treatment. The aim of this study was to reconcile these two apparently incompatible observations. We examined events near the root tip before and after galactose treatment since the tip region is the site of elongation and of high carbon deposition in the root. The treatment rapidly decreased root extension along the whole growing zone. In contrast, turgor pressure, measured directly with the pressure probe in the cortical cells of the growing zone, rapidly increased by 0.15 MPa within the first hour following treatment, and the increase was maintained over the following 24 h. Both tensiometric measurements and a comparison of turgor pressure with local growth rate demonstrated that a rapid tightening of the cell wall caused the reduction in growth. Single cell sampling showed cell osmotic pressure increased by 0.3 MPa owing to accumulation of both organic and inorganic solutes. The corresponding change in cell water potential was a rise from -0.18 MPa to approximately zero. More mature cells at 14 mm from the root tip (just outside the growing region) showed a qualitatively similar response.Galactose treatment rapidly increased the import of recently fixed carbon (RFC) into the whole root as deduced by C-11 labelling of photoassimilate. In contrast, there was a significant decrease in import of recently fixed carbon into the apical 5 mm concomitant with the increase in turgor in this region. No decrease in import of recently fixed carbon was observed 5-15 mm from the root tip despite the increase in cortical cell turgor. These data are consistent with direct symplastic connections between the growing cells and the phloem supplying the solutes in the apical, but not the basal, regions of the growing zone. Hence, the inhibition of growth and the elevation of solute import induced by galactose are spatially separated within the root